Context: Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic insulin resistance (IR) and glucose intolerance. Indian data in this area are sparse, and hence, we undertook this study to find the relationship between NAFLD and diabetes. Objective: The objective of this study is to find the relationship between NAFLD and various levels of glucose intolerance and IR. Settings and Design: A cross-sectional study on 150 patients aged 18 or above and diagnosed to have NAFLD by ultrasonography from October 2013 to June 2015 who were admitted or outpatients in the Department of General Medicine, Kasturba Medical College, Manipal. Methodology: NAFLD was graded as mild, moderate or severe by a single radiologist using high-resolution B-mode ultrasonography system. Basic anthropometric, clinical examination and relevant biochemical investigations were done for all patients. Glycated haemoglobin (HbA1c), fasting plasma glucose (FPG) and post-load plasma glucose (PG) were measured in all subjects, fasting insulin levels were measured in all subjects by chemiluminescence method to determine IR and it was calculated using homeostasis model assessment model. Statistical Analysis Used: Data were analysed using SPSS software version 15 and P < 0.05 considered as statistically significant. Results: The percentage of pre-diabetes and diabetes in NAFLD patients were 52.7% and 30.7% respectively, There was a greater proportion of pre-diabetics and diabetics in the higher grades of NAFLD (P < 0.001). We observed mean FPG, PG and HbA1c levels increased as the severity of NAFLD increased in this study. The overall mean HOMA-IR score in our subjects was found to be 4.68 ± 2.81 and 76.7% of patients had IR was significantly (P < 0.001) higher in Grade 2 and Grade 3 NAFLD. Conclusions: There is a high prevalence of pre-diabetes and diabetes in patients with NAFLD, and this is associated with increasing IR in higher grades of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) which is characterised by an accumulation of fat in the liver with little or no alcohol consumption is the most common cause of liver disease worldwide.[1] The prevalence estimates of NAFLD ranges from 25% to 45% in most studies.[2] The community prevalence of NAFLD in India varies from 5% to 28%.[3],[4],[5] Mohan et al. found 32% prevalence of NAFLD in the urban population of Chennai in 2009 using ultrasonography (USG).[6]

NAFLD is typically associated with central obesity, diabetes, hypertension, dyslipidaemia and metabolic syndrome.[7] Insulin resistance (IR) promotes the fatty acid accumulation and oxidative stress [8] and plays a key role in disease progression in the liver. Further, NAFLD is also known to contribute to glucose intolerance making this a vicious cycle. In India the prevalence of pre-diabetes and diabetes in NAFLD has been found to be variable from 33% to 64%.[6],[9],[10] Hence, we consider analysing the relationship between these two disorders in a south Indian rural population. This study aims to understand the relationship between various grades of NAFLD and level of glucose intolerance as well as IR.

Methodology

This cross-sectional study was initiated after obtaining clearance from the Ethical Committee of Kasturba Medical College (KMC), Manipal, Karnataka, India (IEC No: 508/2013). Patients aged 18 or above and diagnosed to have NAFLD by USG from October 2013 to June 2015 who were admitted or outpatients in the Department of General Medicine, KMC Manipal, after obtaining written informed consent were included. Relevant history regarding alcohol consumption and intake of hepatotoxic drug such as antituberculosis treatment was elicited and excluded. Hepatitis B and C screening was done in all patients to exclude other significant hepatic condition.

With anticipated prevalence of 55% and 20% relative precision with 95% confidence interval, minimum sample size was calculated to be 141.

Ultrasound abdomen was performed by a single radiologist using high-resolution B mode ultrasonography system, Phillips IU 22 Machine, using curvilinear 3–5 MHz transducer and if fatty changes were present, it was graded as mild, moderate or severe by the radiologist.

Relevant history regarding alcohol consumption and other major etiologies of liver disease were asked to exclude patients. Basic anthropometric measures were done for 150 eligible patients. Clinical examination and relevant biochemical investigations were done for all patients. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by ultraviolet with pyridoxal 5'phosphate method using Cobas 6000 auto analyser. Fasting lipid profile was measured including triglycerides (TGs) (GPO grinder method using Roche Cobas E601 analyser machine). The coefficient variation of ALT and AST was 2.36% and 1.50%, respectively; for fasting plasma glucose (FPG) and post-prandial glucose (PPG) were 1.26% and 1.15%, respectively; and for fasting insulin 2.20% and for TG 3.56%.

Height was measured with the patient standing against an upright surface touching it with heels, buttocks and back on a level smooth surface. The measurement was taken when the subject held the breath in inspiration and unit of measurement was meters.

Weight

Subject-wearing minimal clothing was made to stand in the centre of on an electronic weighing machine. Weight was recorded to the nearest tenth of a kilogram. The same machine was used for calculating the weight for all the subjects who underwent the study.

Waist circumference (WC) was measured midway between inferior margin of ribs and the superior border of iliac crest with a plastic tape (WHO steps protocol).[11] WC of ≥90 cm for males and ≥80 cm for females were considered as abnormal (IDF criteria).[12]

Blood pressure recording

Two measurements under standard conditions were made, and the average was taken as the blood pressure (BP). Raised BP was defined as systolic pressure ≥140 mmHg, diastolic ≥90 mmHg or patient on antihypertensive treatment for previously diagnosed hypertension.

Grade 3 fatty liver or severe steatosis: Marked increase in echogenicity with poor visualisation of the posterior structures such as the diaphragm.[15]

Statistical analysis

Data were analysed using SPSS software (SPSS version 15, Chicago, IL, USA). Pearson's Chi-square test was used to study the relation between various categorical variables. Mean (or median) and standard deviation (or 25th percentile, 75th percentiles) values were calculated for continuous variables. One-way ANOVA test was done to study the relation between mean values of the required parameters and various categories of NAFLD.

Results

A total of 150 subjects participated in the study, out of which 76 (50.7%) participants had Grade 1, 55 (36.7%), Grade 2 and 19 (12.7%) and Grade 3 of NAFLD. [Table 1] shows the general characteristics of the study participants. An advanced age, the greater proportion of male subjects, more subjects with obesity and increased WC was observed among higher grades of NAFLD.

The WC of >80 cm in female and 90 cm in male considered abnormal. In this study, 73.6% males were having abnormal WC and 95% females were having abnormal WC and also we observed increase in the number of subjects with abnormal WC as severity of NAFLD increases and it was shown in [Figure 1].

The overall proportion of pre-diabetes and diabetes in NAFLD subjects was observed to be 79 (52.7%) and 46 (30.7%) subjects, respectively. The distribution of glycaemic status of subjects among different grades of NAFLD was shown in [Table 2]. There was a greater proportion of pre-diabetics and diabetics in the higher grades of NAFLD (P < 0.001).

Out of 79 pre-diabetics, isolated IFG were seen in 50 (63.2%) participants, 27 (34.1%) participants had both IFG and IGT and only IGT was observed in 2 (2.5%) participants. It was observed that mean FPG, PPG and HbA1c increased with increased severity of NAFLD. The laboratory profiles of glucose intolerance in different grades of NAFLD are shown in [Table 2].

IR was calculated for the subjects by HOMA-IR with the score above 3 denoting IR. The overall median HOMA-IR score in the study participants was found to be 4.12 (3.06, 5.48) and 114 (76.7%) participants had IR and it was significantly (P < 0.001) higher in Grade 2 and Grade 3 NAFLD as shown in [Table 2].

IR was further categorised as normal (HOMA-IR <3), moderate (HOMA-IR 3–5) and severe (HOMA IR >5).[16] Out of 150 study participants, 36 (24%) of them had normal IR while 60 (40%) had moderate and54 (36%) had severe IR. The number of the subjects with severe IR increased significantly (P < 0.001) as increased in severity of NAFLD. Proportion of subjects with different levels of IR in different grades of NAFLD is shown in [Table 3].

Table 3: Different levels of insulin resistance in different grades of non.alcoholic fatty liver disease

In our study, 88 (58.7%) subjects had dyslipidaemia, and this number was significantly higher (P = 0.001) with higher grades of NAFLD as shown in [Table 1]. 24 (16%) subjects had abnormal AST and 26 (17.3%) had abnormal ALT and these elevations between the three groups were not clinically significant. Hence, these turned out to be a poor indicator of the severity of NAFLD.

Discussion

There is a paucity of data from India on the presence of glucose intolerance in NAFLD patients. Apart from a large epidemiological survey conducted by Mohan et al. which documented the prevalence of NAFLD in patients with glucose intolerance (starting from the opposite end of the spectrum), there is no significant data in this area.

In our hospital-based study, we found more patients with Grade 1 NAFLD than Grade 3. The possible explanations for this being that India may be in the initial stages of NAFLD epidemic or the earlier detection of the disease by USG.

NAFLD was found to be more prevalent in males from Indian and other Asian studies. Mahaling et al. in a study from Maharashtra also came up with similar statistics of male preponderance [17],[18] Male gender is also associated with the more severe disease with histological non-alcoholic steatohepatitis, hepatic fibrosis and higher overall mortality.[19],[20] It was also noted that male patients had severe grades of NAFLD as documented by Mohammadi et al.[21] Female hormones may have a protective effect against the disease and it is more common among post-menopausal women than younger age groups. In our study also, there was a higher percentage of males in Grade 3 NAFLD as compared to Grade 2 and 1.

Obesity was more prevalent in Grade 2 and Grade 3 NAFLD as shown in [Table 1]. Kwon et al.[22] and Abangah et al.[23] also found similar results from other parts of the world. In obese individuals, there is increased the supply of free fatty acid (FFA) to the liver from the diet, adipose tissue and also through de novo lipogenesis promoting hepatic steatosis. Due to excess of circulating FFA, an abundance of pro-inflammatory cytokines with a relative or absolute deficiency of anti-inflammatory cytokines, which is governed by the hormonally active adipose tissue.

Our subjects also had significant visceral fat with higher WC in Grade 2 and Grade 3 NAFLD. Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) have different expression of adipokines and cytokines. SAT has a predominant expression of leptin and adiponectin whereas VAT predominantly secrets tumor-necrosis factor-alpha and interleukin 6 and 8. VAT may contribute more to IR than SAT due to its cytokine profile.

Type 2 diabetes mellitus (T2DM) and NAFLD are pathophysiologically related. This relationship among T2DM, IR and NAFLD are expected because insulin is subsequently delivered directly to the portal vein after secretion, taking the same route as the absorbed glucose, and the liver eliminates a large portion of portal insulin at the first pass.[24] In our study, 83.3% of our subjects had glucose intolerance of which the majority (52.7%) was pre-diabetic. Moreover, 30% of our patients had overt diabetes. This was in accordance with a western study by Ortiz-Lopez et al.[25] in which 85% of the subjects had abnormal glucose metabolism (pre-diabetes and diabetes). Singh et al.[26] in 2013, also demonstrated a higher glucose intolerance in patients with NAFLD.

Further, we found that there is a direct relationship between glucose intolerance and severity of NAFLD with increasing pre-diabetes and diabetes, as we move from Grade 1 to Grade 3 NAFLD, with Grade 3 having a very high percentage of diabetic patients (78.9%) as shown in [Table 2]. By definition, pre-diabetes and diabetes are states of relative insulin deficiency that, when combined with excess caloric intake and obesity, will commonly contribute to increased portal fatty acids and ectopic fat deposition, including in the liver. In addition, the glucose intolerance may significantly increase the hepatic destruction as shown by Haukeland et al.[27] who opined that T2DM and pre-diabetes are the only predictors for development of steatohepatitis and fibrosis in patients with NAFLD.

In our study group, the majority of the subjects had pre-diabetes which included IFG/IGT/IFG andIGT. In patients having IFG alone (63.2%), about half the number (34.1%) had IFG + IGT which was detected after a standard 75 g OGTT, showing the pre-diabetes tendency throughout the glycaemic range. Yun et al.[28] did OGTT in 48 patients with NAFLD and impaired fasting glucose and found 37.6% to have IGT also. Only 2% of our subjects were found to be having isolated IGT without IFG. The pathophysiology of IFG seems to include the following key defects: reduced hepatic insulin sensitivity, beta cell dysfunction and/or chronic low beta cell mass, altered glucagon-like peptide-1 secretion and inappropriately elevated glucagon secretion. This clearly explains the increased proportion of IFG in this study.[29]

NAFLD is considered the hepatic component of the metabolic syndrome and IR represents its pathophysiological hallmark. IR in NAFLD is characterised by reduced whole-body, hepatic and adipose tissue insulin sensitivity. Hepatic steatosis and IR are considered to be the parts of a vicious cycle both being the cause and effect of the other. Moreover, Asian Indians phenotypes have increased WC and visceral fat which in turn makes them more susceptible to IR.[30]

The majority of study participants, specifically 76.7% had IR and the median HOMA-IR score was 4.12 (3.06, 5.48). In a study by Park et al.,[31] 77.8% of the subjects were found to have IR. The two Indian studies which looked for IR in NAFLD were by Bajaj et al.[32] and Bhat et al.[33] and the prevalence of IR in these studies were 46.15% and 97.5%, respectively. We further classified the subjects into three categories-normal IR, moderate IR and severe IR according to Matthews et al.[16] and analysed their proportion in each grade of NAFLD. Proportion of subjects with severe IR progressively increased through the grades of NAFLD as illustrated in [Table 3]. Singh et al. showed IR in 60% of the subjects and he too demonstrated rising IR from 61.8% to 100% in Grades 1–3.[26]

There are some limitations in our study. The diagnosis of fatty liver was based on ultrasonography and not by liver biopsy. It is well known that liver enzymes are poor indicators of NAFLD and hence could be relied upon for diagnosis. Liver biopsy is an invasive procedure with inherent complications which limits its usage in routine clinical practice. USG is non-invasive and well accepted as diagnostic criteria for NAFLD and has been used in other major studies such as by Mohan et al. Hence, we preferred this method of diagnosis. The second limitation could be that since this is a cross-sectional study, temporal or causal relations between NAFLD and glucose intolerance cannot be established. The strength of the study is that the USG was interpreted by a single trained radiologist in a tertiary care setting. This translated into fewer errors in the grading of NAFLD in our study group. Further, all patients with glucose intolerance underwent OGTT to clearly define their glycaemic status, and this helped us to classify them correctly.

Conclusion

This study has important clinical implications. Given the high proportion of pre-diabetes and diabetes in patients with NAFLD, it is mandatory to screen patients with NAFLD for hyperglycaemia. An early diagnosis of diabetes in this high-risk group of patients would provide an opportunity to direct therapy toward IR and optimal glycaemic control to create a favourable 'metabolic memory' early in the disease. That would reduce the subsequent risk of diabetes complications specifically and also perhaps liver complications such as progression to cirrhosis.[34]

Chalasani N, Younossi Z, Lavine JE, Diehl AM, Brunt EM, Cusi K, et al. The diagnosis and management of non-alcoholic fatty liver disease: Practice guideline by the American Association for the Study of Liver Diseases, American College of Gastroenterology, and the American Gastroenterological Association. Hepatology 2012;55:2005-23.

World Health Organization, Western Pacific Region, The Asia Pacific Perspective. Redefining Obesity and its Treatment, World Health Organization, International Associaton for the Study of Obesity and International Obesity Task Force. Melbourne: International Diabetes Institute; 2000.